The broad, long-term objectives of this project are to identify, characterize, and exploit targets for new safe and effective antiviral agents against the human pathogens, herpes simplex virus (HSV) and human cytomegalovirus (CMV). The project takes a combined approach to drug discovery in which drug targets are identified by mapping mutations conferring drug resistance, shedding light on drug mechanisms. The targets are characterized using molecular techniques. The results are used to discover new drugs and gain biological and biochemical insights. The first specific aim is to characterize the interactions of HSV DNA replication proteins and discover and characterize drugs that disrupt them. The mechanisms of a new small molecule drug that inhibits the subunit interactions of HSV DNA polymerase and has antiviral activity will be studied both in vitro and in infected cells. New drugs, including ones derived by structure-based design will be tested. Interactions among other HSV DNA replication proteins will be mapped and their importance assessed by analysis of mutants. Structures will be investigated by X-ray crystallography, and exploited for structure-based drug design. New drugs will be identified using high throughput screening (HTS) and tested using functional assays that reflect the importance of protein-protein interactions, and for antiviral activity. The second specific aim investigates the CMV UL97 protein kinase. HTS will be used to discover new drugs that inhibit this enzyme and they will be tested for antiviral activity and mechanisms. The structure of UL97 will be investigated by X-ray crystalography, and exploited for structure-based drug design. The role of UL97 in nuclear egress will be explored using viral mutants, identification of the natural substrates of this enzyme that are involved in this process, and light and electron microscopic approaches. The third specific aim is to identify new herpesvirus drug targets by studying resistance to drugs with unknown mechanisms, and to use HTS to discover drugs that inhibit stages of viral replication for which few inhibitors exist.